1. Field of the Invention
This invention relates to proteins, polypeptides, or fragments thereof, which interact with autoantigens of autoimmune diseases, such as type I diabetes. More particularly the invention relates to polypeptides, or fragments thereof, which interact with human ICA512 and phogrin. The invention also relates to nucleic acid sequences which encode these polypeptide or polypeptide fragments. The invention also relates to methods for identifying and treating individuals who suffer from or are susceptible to diabetes, screening for autoimmune diseases such as type I diabetes, and modulating hormone and neuropeptide secretion using proteins or protein fragments which interact with autoantigens of autoimmune diseases.
2. Description of the Related Art
Insulin dependent diabetes mellitus (also known as IDDM or type I diabetes) primarily afflicts young people. Although insulin is available for treatment, the several-fold increased morbidity and mortality associated with this disease urge the development of early diagnostic and preventive methods. The destruction of pancreatic .beta.-cells, which precedes the clinical onset of IDDM, is mediated by autoimmune mechanisms. Among the most thoroughly studied autoimmune abnormalities associated with the disease is the high increase of circulating .beta.-cell-specific autoantibodies at the time of diagnosis.
A major goal of diabetes research has been to develop immune interventions that block or inhibit the destruction of .beta.-cells and development of IDDM. For example, U.S. Pat. No. 5,512,447, the disclosure of which is incorporated herein by reference, describes assays for the detection of diabetes and prediabetic status by exposing patient serum samples to purified ligand capable of binding autoantibodies specific for a 64 kD autoantigen present in pancreatic .beta.-cells. One of the most intriguing observations resulting from studying the cell biology of .beta.-cell autoantigens is that all major targets of IDDM autoantibodies identified thus far are directly connected with the secretory apparatus of .beta.-cells (Solimena, Diabetes, Metab. Rev. 14:227-240 (1994)).
A large body of evidence indicates that protein and lipid phosphorylation participates in the trafficking of secretory granules (SGs), synaptic vesicles (SVs), and synaptic-like microvesicles (SLMVs) of neuroendocrine cells, including pancreatic .beta.-cells. Several membrane and cytosolic phosphoproteins involved in priming, docking and fusion of regulated secretory vesicles (Ferro-Novick, S. et al., Nature 370:191-193 (1994); Martin, T. F., Curr. Opin. Neurobiol. 4:626-632 (1994); Calakos, N. et al., Physiol. Rev. 76:1-29 (1996)) have been shown to be substrates for serine/threonine phosphorylation (Greengard, P. et al., Science 259:780-785 (1993); Ashcroft, F. M. et al., J. Cell Biochem. 55 Supp:54-65 (1994)). However, less evidence is available implicating tyrosine phosphorylation in these processes. The following evidence suggests that tyrosine phosphorylation may play a role in regulated secretion.
The protein tyrosine kinase (PTK) inhibitors genistein and herbimycin A both stimulate insulin secretion in neonatal islets (Sorenson, R. L. et al., Endocrinol. 134:1975-1978 (1994)), and genistein has been shown to affect the ATP-dependent priming of SGs in semi-intact PC12 cells (Hay, J. C. et al., J. Cell Biol. 119:139-151 (1992)). In GH3 cells, both PTK and protein tyrosine phosphatase (PTP) inhibitors have been shown to impair the biogenesis of SGs from the trans-Golgi network (Austin, C. D. et al., J. Cell Biol. 135:1471-1483 (1996)) as well as modulate the activity of L-type Ca.sup.2+ channels (Cataldi, M. et al., J. Biol. Chem. 271:9441-9446 (1996)) which are known to be coupled to neurosecretion.
Synaptophysin and synaptogyrin, two intrinsic membrane proteins of SVs, have both been shown to be tyrosine phosphorylated (Pang, D. T. et al., Proc. Natl. Acad. Sci. U.S.A. 85:762-766 (1988); Stenius, K., et al., J. Cell Biol. 131:1801-1809 (1995)), but the physiological relevance has not yet been determined. Furthermore, PTK pp60.sup.c-src has been shown to be peripherally associated with regulated secretory vesicles, including SGs of chromaffin cells (Parsons, S. J. et al., Biochem. Biophys. Res. Comm. 134:736-742 (1986); Grandori, C. et al., J. Cell Biol. 107:2125-2135 (1988)).
Annexins (soluble Ca.sup.2+ and phospholipid-binding proteins) have been implicated in exocytosis in different cell types, including endocrine cells (Burgoyne, R. D. et al., J. Anat. 183:309-314 (1993)). Annexin II, in particular, has been shown to bind chromaffin SGs and to reconstitute secretion from permeabilized chromaffin cells of the adrenal medulla (Sarafian, T. et al., C.J. Cell Biol. 114:1135-1147 (1991)), and has been shown to be tyrosine phosphorylated by 60.sup.c-src (Hubaishy, I. et al., Biochemistry 34:14527-14534 (1995)). pp60.sup.c-src has also been shown to phosphorylate botulinum A and E (Ferrer-Montiel, A. V. et al., J. Biol. Chem. 271:18322-18325 (1996)), two clostridium neurotoxins which cause the block of neurosecretion and paralysis by cleaving the synaptobrevin binding protein SNAP-25 at neuromuscular junctions (Tonello, F. et al., Adv. Exp. Med. Biol. 389:251-260 (1996)). Since tyrosine phosphorylation enhances the protease activity of both botulinum A and E, it is possible that tyrosine phosphorylation and regulated secretion are coupled in certain cells.
Islet cell autoantigen 512 (ICA512, also known as IA-2 and PTP35), is an intrinsic membrane protein of SGs which is expressed in virtually all neuroendocrine cells including peptide-secreting endocrine cells, neurons of the autonomic nervous system, and neurons of the hypothalamus and the amygdala in the brain (Solimena, M. et al., EMBO J. 15:2102-2114 (1996)). Human ICA512 has been identified as an autoantigen of IDDM (Rabin, D. U. et al., Diabetes 41:183-186 (1992); Rabin, D. U. et al., J. Immunol. 152:3183-3188 (1994); Lan, M. S. et al., DNA Cell Biol. 13:505-514 (1994)), and has been postulated to play a role in regulated peptidergic secretion from neuroendocrine cells, including insulin-secreting cells of the pancreatic islets. The open reading frame of human ICA512 encodes a protein of 979 amino acids (FIG. 1). The sequence includes a signal peptide (residues 1-34) and two putative N-glycosylation sites (residues 506 and 524) in the extracellular domain (residues 1-575, henceforth defined as ectodomain), a single transmembrane domain (residues 576-600), and a cytoplasmic domain (residues 601-979) which includes a region displaying homology to protein tyrosine phosphatases (PTPs) (residues 696-979). Like several other enzymatically active receptor protein tyrosine phosphatases (RPTPs) (Streuli, M. et al., EMBO J. 11:897-907 (1992); Serra-Pages, C. et al., J. Biol. Chem. 269:23632-23641 (1994); Brady-Kalnay, S. M. et al., J. Biol. Chem. 269:28472-28477 (1994); Pulido, R. et al, Proc. Natl. Acad. Sci. U.S.A. 92:11686-11690 (1995)), ICA512 is processed within its ectodomain (Solimena, M. et al., EMBO J. 15:2102-2014 (1996); Hermel et al., Eur. J. Neurosci. 11:20690 (1999)). Cleavage of ICA512 (arrow in FIG. 1) generates a 65 kD transmembrane fragment (residues 449-979) which remains associated with SG membranes, and a putative N-terminal fragment (residues 35-448).
The cytoplasmic domain of ICA512 is homologous with PTPs and suggests that ICA512 participates in signal transduction pathways involving tyrosine phosphorylation. Several features, however, distinguish ICA512 from conventional RPTPs.
First, the ectodomain of human ICA512 does not contain any of the motifs found in most RPTPs, including Ig domains, type III fibronectin repeats, MAM (meprin, A5, PTP.mu.) domains, or carbonic anhydrase-like motifs, all of which are thought to mediate cell--cell or cell-matrix contact (Brady-Kalnay, S. M. et al., Curr. Opin. Cell Biol. 7:650-657 (1995); Streuli, M. Curr. Opin. Cell Bio. 8:182-188 (1996)). (The RGD motif of the type III fibronectin repeat, however, is present once in the ectodomains of rat and mouse ICA512 (Passini, N. et al., Proc. Natl. Acad. Sci. U.S.A. 92:9412-9416 (1995); Lu, J. et al., Biochem. Biophys. Res. Comm. 204:930-936)).
Second, ICA512 joins phogrin and striate enriched phosphatase (STEP) isoforms as a member of the RPTP family which resides in an intracellular compartment (Rydelk, F. L. et al. Soc. Neurosci. 22:1005 (400.5) (1996); Bult A., et al., J. Neuroscience 16:7821-7831 (1996)). In contrast, the majority of other receptor protein tyrosine phosphatases are believed to be constitutively expressed at the plasma membrane. Phogrin (also known as IA-2.beta. or ICAAR), in particular, is a mammalian protein with significant structural and most likely functional homology to ICA512 (Wasmeier, C. et al., J. Biol. Chem. 271:18161-18170 (1996)). Phogrin is also associated with secretory granules of neuroendocrine cells, including .beta.-cells, and is an autoantigen of IDDM (Pietropaolo, M. et al., Diabetes Care 20:208-214 (1997); Hatfield, E. C., Diabetologia 40:1327-1333 (1997)).
Third, recombinant ICA512 does not display PTP activity when tested with common PTP substrates (Rabin, D. U. et al., Diabetes 41:183-186 (1992); Lan, M. S. et al., DNA Cell Biol. 13:505-514 (1994); Lu, J. et al., Biochem. Biophys. Res. Com. 204:930-936 (1994)). The lack of catalytic activity of ICA512 results from two amino acid substitutions within its PTP homology domain: an aspartic acid (D) instead of an alanine (A) at position 911 within the so-called PTP "signature motif", and an alanine instead of an aspartic acid at position 877. However, an ICA512 mutant in which alanine 877 and aspartic acid 911 have been replaced with aspartic acid and alanine, respectively, displays PTP activity similar to conventional PTPs (Magistrelli, G. et al., Biochem. Biophys. Res. Comm. 227:581-588 (1996)). In addition, replacement of aspartic acid with alanine within the PTP signature motif of phogrin is sufficient to confer PTP activity (Rydelk, F. L. et al. Soc. Neurosci. 22:1005 (400.5) (1996)).
There is evidence that other members of the RPTP family interact with proteins of the spectrin family. CD45, a receptor involved in T- and B-cells proliferation, has been shown to bind .beta.II-spectrin (also known as .beta.-fodrin) via its C-terminus PTP domain (Iida et al., J. Biol. Chem. 269:28576-28583 (1994)). Strikingly, this PTP domain in CD45, like that in ICA512, contains an aspartic acid rather than an alanine within its PTP signature motif and appears to be enzymatically inactive. A protein termed TRIO, which contains multiple spectrin repeats, has been recently found to interact with the regulatory, enzymatically inactive, PTP domain of RPTP LAR (Debant, A. et al., Proc. Natl. Acad. Sci. U.S.A. 93:5466-5471 (1996)). Taken together, this data suggests that modified, non-catalytic PTP domains may mediate the interaction of RPTPs with the actin cytoskeleton via an interaction with different members of the spectrin family.
There is also evidence that spectrins can be the targets of autoimmunity in human diseases. For instance .alpha.II-spectrin (also known as .alpha.-fodrin) has been recently found to be a major autoantigen in Sjorgren syndrome (Haneji, N. et al., Science 276:604-607 (1997); Yanagi et al., Eur. J. Immunol. 28:3336 (1998)), a disorder resulting from the autoimmune infiltration of the salivary gland. Recently, both cellular and humoral autoimmunity directed against .alpha.II-spectrin has been detected in the non-obese diabetic (NOD) mouse (Yanagi et al., Eur. J. Immunol. 28:3336-3345 (1998)), a mouse strain that spontaneously develops an autoimmune sialadenitis resembling human Sjogren syndrome as well as an autoimmune diabetes resembling human type I diabetes. These data raise the question of whether autoimmunity directed against .alpha.II-spectrin can develop in type I diabetic patients in the absence of Sjogren syndrome. Relevant to this question is whether .alpha.II-spectrin is expressed in pancreatic .beta.-cells. To the inventor's knowledge, no members of the spectrin family, including .alpha.II-spectrin, has been reported to be expressed in pancreatic .beta.-cells.
Heretofore, studies on vesicular trafficking in neuroendocrine cells and signal transduction via tyrosine phosphorylation have proceeded without significant overlap. Recently, however, it has been postulated that a close relationship may exist between the cell biology of regulated secretion and the organization of the cortical cytomatrix, a preferential target for tyrosine phosphorylation. ICA512 and phogrin could each play a role in regulating peptide hormone secretion and/or survival and differentiation of neuroendocrine cells. However, the precise role of these autoantigens remains elusive, as the proteins appear to lack PTP activity. Therefore, there is a need in the diabetes treatment art for the identification, isolation, and characterization of protein molecules, or fragments thereof, that associate with autoantigens of type I diabetes, such as human ICA512 and phogrin, in order to assist in the determination of the precise role of these autoantigens in peptide hormone secretion. In addition, such proteins, or fragments thereof, could be the targets of autoimmunity in human diseases related to peptide hormone secretion, including IDDM, and hence would be useful as a tool in attenuating or screening for such diseases. The present invention is believed to be an answer to that need.